Nanostructured microelectrodes and biosensing devices incorporating the same

ABSTRACT

Nanostructured microelectrodes and biosensing devices incorporating the same are disclosed herein.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.13/061,465 which is a United States National Stage Application filingunder 35 U.S.C. §371 of International Application No. PCT/CA2009/001212,filed on Sep. 1, 2009, which claims the benefit of priority of U.S.Provisional Patent Application Ser. No. 61/093,667, filed on Sep. 2,2008, entitled Nanostructured Microelectrodes and Biosensing DevicesIncorporating the Same, now expired, the contents of each of which ishereby incorporated by reference herein in its entirety. InternationalApplication No. PCT/CA2009/001212 was published under PCT Article 21(2)in English.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically as a text file in ASCII format and is herebyincorporated by reference in its entirety. Said text file, created onFeb. 20, 2015, is named 109904-0004-302.txt and is 6,000 bytes in size.

BACKGROUND OF THE INVENTION

Genomic analysis is revolutionizing early disease diagnosis anddramatically enhancing patient care (McGuire et al. Science 317:1687,Srinivas et al., Lancet Oncol. 2: 698). Microarrays (Drmanac et al.,Science 260:1649, Hacia et al., Nat. Genet. 14:441) and polymerase chainreaction (PCR)-based techniques (Saiki et al., Science 230:1350) have,as tools, helped to spearhead this revolution, enabling the discoveryand the initial development of assays for patient testing (Morris etal., Curr. Opin. Oncol. 19:547). However, spreading the reach of thegenomics revolution to the patient bedside demands cost effective toolsfor individual biomarker profiling assessed relative to a positeddisease state. Specifically, tools enabling routine patient carepreferably would be simpler, more portable, and less expensive thanPCR-based methods, yet should retain a high degree of selectivity andsensitivity.

Biomarker analysis based on electronic readout has long been cited as apromising approach that would enable a new family of chip-based deviceswith appropriate cost and sensitivity for medical testing (Drummond etal., Nat. Biotechnol. 21:1192, Katz et al., Electroanalysis 15: 913).The sensitivity of electronic readout is in principle sufficient toallow direct detection of small numbers of analyte molecules with simpleinstrumentation. However, despite tremendous advances in this area aswell as related fields working towards new diagnostics (Clack et al.,Nat. Biotechnol. 26:825, Geiss et al., Nat. Biotechnol. 2:317, Hahm etal., Nano Lett. 4:51, Munge et al., Anal. Chem. 77:4662, Nicewamer-Penaet al., Science 294:137, Park et al., Science 295:1503, Sinensky et al.,Nat. Nano. 2:653, Steemers et al., Nat. Biotechnol. 18:91, Xiao et al.,J. Am. Chem. Soc. 129:11896, Zhang et al., Nat. Nano. 1:214, Zhang etal., Anal. Chem. 76:4093, Yi et al., Biosens. Bioelectron. 20:1320, Keet al., Science 319:180, Armani et al., Science 317:783), currentmultiplexed chips have yet to achieve direct electronic detection ofbiomarkers in cellular and clinical samples. The challenges that havelimited the implementation of such devices primarily stem from thedifficulty of obtaining very low detection limits in the presence ofhigh background noise levels present when complex biological samples areassayed, and the challenge of generating multiplexed systems that arehighly sensitive and specific.

The miniaturization of electrochemical systems continues to be a majorfocus in analytical and bioanalytical chemistry (Matysik,Miniaturization of Electroanalytical Systems (Springer-Verlag, 2002)),as the attainment of enhanced sensitivity may be enabled with systemspossessing micro-to nano-scale dimensions (Szamocki et al., A. Anal.Chem. 2007, 79, 533-539). A great deal of work has been carried out withelectrodes with dimensions on the micrometer or sub-micrometer scale.These systems offer many advantages over conventional macroelectrodessuch as faster double-layer charging, reduced ohmic loss, highmass-transport rates, and high current density (Bond et al. Anal. Chimi.Acta 1989m 216, 177-230, Heinze, Angew. Chem. Int. Ed. 2003, 32,1268-1288). Indeed, such electrodes have become well-established toolsin a wide range of analytical applications (Bard, ElectrochemicalMethods: Fundamentals and Applications (Wiley, New York, 2001), Reimers,Chem. Rev. 2007, 107, 590-600, Zosic, Handbook of electrochemistry(Elsevier, 2007)). Working with nanoscale electrodes, however, issignificantly more challenging, as fabrication is typicallylabour-intensive, insufficiently reproducible, and the currents obtainedfrom such structures are typically difficult to measure accurately.

The use of nanowire electrodes for ultrasensitive nucleic acids andprotein detection has been investigated (Gasparac et al. J Am Chem Soc726:12270). The use of this electrode platform enables theelectrochemical detection of picomolar levels of analytes, a level ofsensitivity that is not possible using macroscale materials. Although ithas been reported that nanowires are able to detect attomolar levels ofanalytes, this actually corresponds to picomolar levels when dealingwith the volumes typically used for analysis. It has also beendemonstrated that nanoparticle-modified electrodes may exhibit severaladvantages over conventional macroelectrodes such as enhancement of masstransport, catalysis, high effective surface area and control overelectrode microenvironment (Katz et al. Electroanalysis 2004, 16, 19-44,Welch et al. Anal. Bioanal. Chem. 2006, 384, 601-619). Manufacturingarrays of nanowire electrodes, however, is non-trivial.

Boron doped diamond microelectrodes modified by electrodeposition ofplatinum nanoparticles have been used for the oxidative determination ofAs(III) at levels below 1 ppb (Hrapovic et al. Anal. Chem. 2007, 79,500-507). However, this type of electrode cannot be incorporated into anarray-based format for multiplexed experiments.

The analysis of panels of nucleic acid or protein biomarkers offersvaluable diagnostic and prognostic information for clinical decisionmaking. Existing methods that offer the specificity and sensitivity toprofile clinical samples are typically costly, slow and serial. There isthus a need for an ultrasensitive device for detecting biomarkers in amultiplexed fashion.

SUMMARY OF THE INVENTION

In one aspect, the invention features nanostructured microelectrodes(NMEs). NMEs are electrodes, which are nanotextured and thus have anincreased surface area. Preferred NMEs are comprised of a noble metal,(e.g. gold, platinum, palladium, silver, osmium, indium, rhodium,ruthenium); alloys of noble metals (e.g. gold-palladium,silver-platinum, etc.); conducting polymers (e.g. polypyrole (PPY));non-noble metals (e.g. copper, nickel, aluminum, tin, titanium, indium,tungsten, platinum); metal oxides (e.g. zinc oxide, tin oxide, nickeloxide, indium tin oxide, titanium oxide, nitrogen-doped titanium oxide(TiOxNy); metal silicides (nickel silicide, platinum silicide); metalnitrides (titanium nitride (TiN), tungsten nitride (WN) or tantalumnitride (TaN)), carbon (nanotubes, fibers, graphene and amorphous) orcombinations of any of the above. NMEs of the above-described materialsare highly conductive and form strong bonds with probes (e.g. nucleicacids and peptides). Preferred NMEs have a height in the range of about0.5 to about 100 microns (pm), for example in the range of about 5 toabout 20 microns (e.g. 10 microns); a diameter in the range of about 1to about 10 microns; and have nanoscale morphology (e.g. are nanostructured on a length scale of about 1 to about 300 nanometers and morepreferably in the range of about 10 to about 20 nanometers). NMEs can beany of a variety of shapes, including hemispherical, irregular (e.g.spiky), cyclical (wire-like) or fractal (e.g. dendritic). The surface ofan NME may be further coated with a material, which maintains theelectrode's high conductivity, but facilitates binding with a probe. Forexample, nitrogen containing NMEs (e.g. TiN, WN or TaN) can bind with anamine functional group of the probe. Similarly, silicon/silica chemistryas part of the NME can bind with a silane or siloxane group on theprobe.

In another aspect, the invention features an NME associated with aprobe. In one embodiment, the probe is a nucleic acid (e.g. aribonucleic acid (RNA), deoxyribonucleic acid (DNA) or analog thereof,including, for example, a peptide nucleic acid (PNA), which contains abackbone comprised of N-(2-aminoethyl)-glycine units linked by peptidesrather than deoxyribose or ribose, peptide nucleic acids, locked nucleicacids, or phosphorodiamidate morpholino oligomers. Under appropriateconditions, the probe can hybridize to a complementary nucleic acid toprovide an indication of the presence of the nucleic acid in the sample.In another embodiment, the probe is a peptide or protein (e.g. antibody)that is able to bind to or otherwise interact with a biomarker target(e.g. receptor or ligand) to provide an indication of the presence ofthe ligand or receptor in the sample. The probe may include a functionalgroup (e.g., thiol, dithiol, amine, carboxylic acid) that facilitatesbinding with an NME. Probes may also contain other features, such aslongitudinal spacers, double-stranded and/or single-stranded regions,polyT linkers, double stranded duplexes as rigid linkers and PEGspacers.

In a further aspect, the invention features a plurality of NMEs arrayedon a substrate. Preferred substrates are comprised of a semiconductormaterial, such as silicon, silica, quartz, germanium, gallium arsenide,silicon carbide and indium compounds (e.g. indium arsenide, indium,antimonide and indium phosphide), selenium sulfide, ceramic, glass,plastic, polycarbonate or other polymer or combinations of any of theabove. Substrates may optionally include a passivation layer, which iscomprised of a material, which offers high resistance and maintains asmall active surface area. Examples of appropriate materials include:silicon dioxide, silicon nitride, nitrogen doped silicon oxide (SiOxNy)or paralyene. In certain embodiments, the plurality of NMEs arrayed onthe substrate include probes in conjunction with monolayer spacers,which minimize probe density, thereby maximizing complexationefficiency. Preferred monolayer spacers have an affinity to metal andcan be comprised, for example, of a thiol alcohol, such asmercaptohexanol, alkanethiols, cysteine, cystamine, thiol-amines,aromatic thiols (e.g. benzene thiol, dithiol), phosphonic acids orphosphinic acids.

Another aspect features biosensing devices, such as integrated circuits,comprising, for example, a substrate; an electrically conductive lead onthe substrate; an insulating or passivation layer covering the lead, theinsulating layer having an aperture exposing a portion of the lead; anda nanostructured microelectrode in electrical communication with theexposed portion of the lead, the microelectrode being adapted togenerate a charge in response to a biomolecular stimulus (e.g. nucleicacid hybridization or protein-to-protein binding.

In still another aspect, the invention features methods formanufacturing NMEs. The use of electrodeposition to grow nano structuredmicroelectrodes from an NME seed allows the sizes and morphologies ofthese structures to be finely controlled, and versatile fabrication ofelectrodes composed of one or a variety of substances. NMEs may beprepared on a biosensing device, such as a chip-based format, such thata series of NMEs may be made on a single chip to enable multiplexedexperiments. This NME system may be particularly useful and versatile,allowing adjusting of several parameters, including: the microscalecontrol of the NME size and shape, the nanoscale control of NMEnanotexturing, and selection of the NME material.

Yet another aspect features methods for manufacturing biosensing deviceshaving nanostructured microelectrodes. For example, the methods cancomprise the steps of providing a substrate and an electricallyconductive lead on the substrate, the lead being covered by aninsulating layer; etching an aperture in the insulating layer to exposea portion of the lead; and electrodepositing an electrically conductivematerial on the exposed portion of the lead to form a nano structuredmicroelectrode as described above.

In another aspect, there is provided a biosensing cartridge comprising:a sample chamber for containing a biological sample; a biosensingchamber for containing a biosensing device as described above andcarrying out a biosensing process.

In yet another aspect, there is provided a biosensing workstationcomprising: a cartridge holder for holding a biosensing cartridge asdescribed above; an instrument tip for accessing the biosensingcartridge; a selection mechanism for selecting a biosensing process tobe carried out; a processor adapted to carry out the biosensing processusing the biosensing cartridge and to determine results of thebiosensing process from electronic signals generated from the biosensingcartridge; and a display for displaying the results of the biosensingprocess.

A further aspect features methods for carrying out a biosensing processusing probe containing nanostructured microelectrodes incorporated intoa device as described above; biasing the microelectrode relative to areference electrode; measuring a reference charge or reference currentflow between the microelectrode and the reference electrode; exposingthe microelectrode to a biomolecular stimulus (e.g. hybridizationbetween a nucleic acid probe with a complementary nucleic acid orbinding between a peptide probe and a binding partner present in abiological sample); measuring a charge or current flow generated at themicroelectrode in response to the biomolecular stimulus; and determiningthe amount of biomolecular stimulus present by comparing the measuredcharge or measured current flow against the reference charge orreference current flow.

NMEs are versatile, robust and easy to work with. In addition, they canbe manufactured using existing silicon CMOS foundry fabricationprocedures for top-metal fabrication, or simple extrapolations thereof,such as electroless deposition or electrodeposition onto top-metallayers from a CMOS foundry, allowing the manufacture of NMEs to beeasily integrated into existing manufacturing facilities. In addition,NMEs are able to consistently attach to probe molecules. Further, NMEspromote ready accessibility of target molecules such that, when a targetmolecule that is complementary to the NME-attached probe molecule entersinto proximity with that probe, hybridization or protein-to-proteinbinding occurs with high probability. NMEs are further compatible withthe performance of electrocatalytic electrochemistry employed in theread-out of the hybridization event.

Other features and advantages of the inventions disclosed herein willbecome apparent from the following detailed description and claims.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1(A) is a schematic of an NME biosensing device; FIG. 1(B) is aschematic cross-sectional view of an aperture for forming an NME; andFIG. 1(C) is a schematic view of the formation of an NME in an aperture;

FIG. 2(A) is a schematic of an NME biosensing device; FIG. 2(B) showsSEM images of NMEs.

FIG. 3 is a schematic of an NME with probes, further showing thepresence of spacers in the probe monolayer and between the electrode andthe probe;

FIG. 4 are SEM images of NMEs with increasing degrees ofnanostructuring;

FIGS. 5A and 5B illustrate steps involved in the sensing of specificsequences using an NME;

FIG. 6 is a cross-sectional view of an integrated circuit having an NME;

FIG. 7 is a circuit diagram of a circuit that may be used with an NME;

FIG. 8 is an illustration of a biosensing device having an array ofNMEs;

FIG. 9 is an illustration of a biosensing device having different NMEs;

FIG. 10 is a schematic illustration of a biosensing cartridge; and

FIG. 11 is a schematic illustration of a biosensing workstation.

DETAILED DESCRIPTION OF THE INVENTION

The present disclosure describes nanostructured microelectrodes (NMEs),which may be used in a biosensing device, such as a biosensing chip.

NMEs

FIG. 1A shows a schematic diagram of an exemplary device incorporatingNMEs. In the example shown, the device is a chip having an array ofeight leads. In this example, the NME is formed on gold leads that taperto a width of 5 microns. The lead is provided on a substrate of Si andSiO₂, although other suitable substrate materials may be used. On top ofthe lead, an insulating layer, such as SiO₂, is deposited toelectrically insulate and passivate the lead. An aperture, in this casea 500 nm hole, is created in the insulating layer to expose a portion ofthe lead.

FIG. 1B is a side view of the portion of the chip where the aperture islocated, showing the layers of the device. Most commonly knownphotolithography techniques may be suitable for creating an aperture,such as 100 nm to 1 μm diameter apertures. This is typically achievablein existing fabrication facilities with high robustness andreproducibility. Given that only this exposed surface iselectrochemically active, electrodeposition (Menke et al. Nature Mater.2006, 5, 914-919) may be used to grow an NME within this space.

FIG. 1C is a cross-sectional schematic of an example of NME deposition,using Pd for the NME. This process will be described in greater detailbelow.

Reference is now made to FIG. 2, which also illustrates the formation ofan NME on a chip. As in FIG. 1A, small electrodes are provided in situon a chip, with the position and electrical contacting of the NMEsdefined photolithographically. As in FIG. 1, this chip is an 8-foldmultiplexed passive chip. On a silicon substrate, a ˜350 nm thick goldlayer is patterned, using conventional photolithography techniques, toconnect eight 5-μm-wide Au leads to large metal pads for connection tooff-chip instrumentation. A pinhole-free insulating SiO₂ layer isdeposited and patterned to create ˜500 nm openings (e.g., by etching) atthe end of each of the Au leads, to expose a section of the lead. Ametal NME is then plated in this opening using electrodeposition.

The NME may comprise different conductive materials. Some examples ofNMEs have been formed as follows, although variations are possible andwill be described in greater detail below: Branched fractal Pd NMEs weredeposited in an aqueous solution containing 5 mM of H₂PdCl₄ and 0.5 M ofHClO₄ using DC potential amperametry at −250 mV for 15 s. HemisphericalPd NMEs with nanoscale roughness were deposited in an aqueous solutioncontaining 5 mM of H₂PdCl₄ and 0.5 M of HCl using DC potentialamperametry at −100 mV for 300 s. Smooth hemispherical Pd NMEs werefabricated in the same solution at 0 mV for 300 s. Au NMEs werefabricated in a gold bath containing 0.01 M solution of HAuCl₄ and 0.5 MH₂SO₄ at −100 mV for 40 seconds. Pt NMEs were fabricated in a platinumbath containing 5 mM solution of H₂PtCl₆ and 0.5 M H₂SO₄ at −100 mV for500 s. NME size and morphology can be controlled by varying the metalsalt concentration, type and concentration of the supportingelectrolyte, and electrodeposition potential and duration.

FIG. 3 is a schematic of an NME with probes with spacers in the probemonolayer and between the electrode and probe. A chemical solutioncontaining a metal cation can be brought into communication with thesurface of the NME and a reference electrode. The reference electrodemay be an NME or a conventional electrode on same lead. An electricalbias can be provided between the NME and the reference electrode. Thechemical solution can then be removed and the electrodes washed. Asolution containing the probe molecule can then be brought intocommunication with the surface of the NME. The probe molecule may bemodified or functionalized so that it binds to the surface of the NME.For example, the probe molecule may be functionalized with a thiol,amine, or carboxylate group.

NMEs with increasing nanostructuring are shown in FIG. 4. Unexpectedly,variation of electrodeposition conditions allowed growth of extremelysmooth hemispherical microelectrodes (left); highly branched nanoscalefractal structures (right); or hemispheres with nanoscale roughness(center). The structure on the left was made with HCl as a supportingelectrolyte with an applied potential of 0 mV. The center structureswere also made with HCl as a supporting electrolyte but with an appliedpotential of −100 mV. The structure on the right was made with HClO₄ asa supporting electrolyte and an applied potential of −250 mV. The scalebar on the figure corresponds to 5 μm unless otherwise indicated.

FIGS. 5A and 5B illustrate steps involved in the sensing of specificsequences (Lapierre et al., Anal. Chem. 75:6321, Ratilainen et al.,Biochemistry 39:7781, Tomlins et al., Science 3/0: 644) In this example,Pd NMEs are first modified with thiol-derivatized probe sequences, andthen target sequences are hybridized. The presence of the target is thentransduced using an electrocatalytic reporter system. Electrocatalysisprovides electronic amplification, or gain, facilitatinghigh-sensitivity readout: hundreds of electrons may result from eachbiomolecular complexation event. The approach used herein relies on theprimary electron acceptor Ru(NH₃)₆ ₃₊, which is electrostaticallyattracted to the electrode surfaces at levels that are correlated withthe amount of bound nucleic acid. The inclusion of Fe(CN)⁶ ³⁻ duringelectrochemical readout serves to regenerate the Ru(III) substrate, asthe Fe(III) species is even easier to reduce, but it iselectrostatically repelled from the electrode and thus only undergoeschemical reduction by Ru(II). This method is also label-free and doesnot require the sample to be processed in any way.

The biosensing device may be provided in the form of a chip, such as anintegrated circuit (IC) chip. In general, an IC incorporating the NMEmay have a substrate with an electrically conductive lead that iscovered by an insulating layer. The insulating layer has an aperturethat exposes a portion of the lead, and the NME is provided at theexposed portion of the lead. The NME is responsive to a biomolecularstimulus. In particular, the NME may be functionalized with probemolecules that undergo a hybridization reaction with a targetbiomolecule (e.g., a nucleic acid sequence), resulting in a chargegenerated at the NME. The IC also has a charge storage (e.g., acapacitor or a battery) in electrical communication with the lead tostore this generated charge. In typical usage, the NME may be exposed toa sample for a known time duration or an integration period, and thecharge stored over that time would then be indicative of the presenceand/or amount of the target biomolecule.

The stored charge may be communicated to a computing device foranalysis, or may be displayed (e.g., through a digital displaycomponent) for direct reading of the charge stored after the integrationperiod.

Such an IC may be manufactured using common IC manufacturing equipment,allowing this device to be easily manufactured and to be less costlythan other forms of biosensing microelectrodes. The materials used maybe those already commonly used in IC manufacturing. For example, thesubstrate may be made from silicon, quartz, glass, ceramics, silica,sapphire, gallium arsenide, or other materials currently used for ICs.The substrates or supports can incorporate conductive material to serveas an electrode. Conductive supports with a gold surface may also beused. The supports usually comprise a flat (planar) surface, or at leasta structure in which the probes or p to be interrogated are inapproximately the same plane. The support can be an electrode, or can beattached to an electrode.

The lead may be made of Au, Al, W, TiN, polysilicon or other commonlyused lead materials. The IC may include a transistor, such as afield-effect transistor (FET) including n-type silicon channel FETs andp-type silicon channel FETs, or a bipolar transistor including n-p-nbipolar junction transistors and p-n-p bipolar junction transistors.

The IC may be provided with, immersed in or otherwise exposed to anelectrocatalytic solution in chemical and electrical communication withthe NME. This may assist in charge generation in the NME.

Reference is now made to FIG. 6. This figure shows a cross-section of anintegrated circuit suited to sensing the presence of biomolecules in abiological sample. The substrate (1) is a conventional semiconductordevice substrate such as silicon. The channel of a transistor (2), agate oxide (3), and a polysilicon gate electrode (4) are shown toillustrate the use of conventional CMOS electronics to form theintegrated circuit's transistors. A metal (5) is used to contact thegate electrode. A passivation oxide (6) separates the silicon transistorlevels below from the top surface of the chip above. A series of metalvials (7) and interconnects provide selective paths for electricalcommunication between the transistor layer and the top electrode(s). Asubstantially planar top surface is a heterogeneous combination of topelectrodes (9) and top insulating material (8). The figure illustratesan NME (10) provided on the electrode, for example using the methodsdescribed above. The figure illustrates probe biomolecules (11) such asthiol-terminated nucleic acids that are displayed for efficienthybridization with complementary target molecules. An electrocatalyticsolution (12) may be employed to provide catalytic read-out ofhybridization with the biomolecules (11). Electrical potentials areconveyed, and currents flowed, in a continuous fashion from the NME (10)through the electrical contacts (9) (7) (5) (4) down to the electroniccircuitry that resides beneath.

Reference is now made to FIG. 7, showing a circuit diagram of an examplecircuit that may be used with the disclosed NME. In this example, thecircuit may provide the following functions: biasing of theprobe-functionalized NME; integration of the current flowing through theNME into a charge store having a known charge-storage capacity; read-outof the voltage on the charge-store; and selection of the charge-store orNME of interest when a two-dimensional array of stores and electrodes isprovided in the context of a highly multiplexed array chip.

The components of the example circuit are now described. A bias voltageis provided at V_(bias); atypical choice of bias may be in the range ofabout 0.1-2.8 V. A bias voltage V_(biasD) is provided at thesource-follower drain; a typical choice may be in the range of about0.1-2.8 V. A bias voltage V_(biasR) is provided at the reset node; atypical choice may be an adjustable value between about −2 V and 2.8 V.The signal voltages are V_(src) which may typically be in the range ofabout 1.5-2.5 V. The column voltage Vent may be in the range of about1.5 V-0.5 V. Timing control signals include that for row select (e.g.,range may be about 0-2.8 V) and for reset (e.g., range may be about 0-4V). The transistors may be the reset transistor Tr(Reset), the read-outbuffer transistor TR(source-follower), and the row-select transistorTR(row-select).

The above biases, signal voltages and timing control signals areexamples only and other values may be used. These biases, voltages andsignals may be selected or adjusted to suit certain applications ormanufacturing conditions, as is commonly known in the art. In thisexample, the probe-functionalized NME may include a NME functionalizedusing a thiolated nucleic acid probe, for example a probe as describedabove. V_(src) is applied to the probe-functionalized electrode andV_(biasR) is applied to a second electrode, which may be a NME or anyother common electrode, in electrical communication with theelectrocatalytic solution. This results in voltage difference betweenthe probe-functionalized electrode and the electrocatalytic solution. Acurrent may thus flow as a consequence of this potential difference. Theamount of current flowing may be typically dependent on the amount ofhybridization on the probe-functionalized NME, that is the current maybe indicative of the amount of target thus detected by the NME.

The operation of the example circuit is now described. In order tocapture the current flowing, I_(sense), the reset transistor is turnedon by setting the node ‘reset’ high enough (e.g., up to 4V, which may bethrough an on-chip charge-pump or regulator circuit as commonly known inthe art) so that node 22 will be charged to a voltage equal to V_(bias)(node 18) which may be typically set to the supply rail: e.g., 2.8V.This is the reset phase. Once this ‘reset’ operation is completed, Node17 may be set to 0V to turn off the reset transistor Tr(Reset) (13). Indoing so, charge injection and parasitic capacitive feedthrough effectswill cause node 22, now becoming a floating node, to drop byapproximately 300 mV. Therefore after the ‘reset’ operation the actual‘reset’ voltage value at node 22 is approximately 2.5V. At this time,the current I_(sense) flowing is dependent on the voltage applied (i.e.,V_(src)−V_(biasR)). With V_(biasR) being able to be set arbitrarily toany voltage level from −2V to 2.8V, the applied potential difference maybe adjusted. The current I_(sense) discharges the parasitic capacitanceat the V_(src) node (22) and its voltage level drops at a rate dependenton the value of the parasitic capacitance at V_(src) (node 22) as wellas the flowing I_(sense) during the integration time. After a specificintegration time, the resulting integrated voltage at node 22 will beread out through transistors TR(source-follower) and TR(row-select), thesource follower buffer transistor and the row-select transistor, bysetting the node SEL (20) to a high level (2.8V).

The charge store that is discharged at node 22 may comprise parasiticcapacitance of one or more of the transistors that are in electricalcommunication with the electrode of the pixel region at V_(src) (node22). The electrode at V_(src) (node 22) may be in electricalcommunication with the gate of a transistor, such as Tr(source-follower)14, which provides a parasitic capacitance. In one example embodiment,the charge store may be provided at least in part by a parasiticcapacitance between the gate and drain of the source followertransistor, Tr(source-follower) 14, and a parasitic capacitance betweenthe source and substrate of the reset transistor, Tr(Reset) 13. Theseare parasitic capacitances between the structures on the semiconductorsubstrate (e.g., the poly, n-well and substrate) on which or in whichthe pixel circuit is formed. In an example embodiment, these parasiticcapacitances may be in the range of about 1-2 femtoFarads or moregenerally in the range of about 0.5 to 3 femtoFarads or any rangesubsumed therein. The contacts to the probe-functionalized NME may beformed in different layers above the regions of the semiconductorsubstrate used to form the transistors. In an alternate embodiment, thepolarity of the bias may be reversed and the parasitic capacitance atV_(src) may be charged instead of discharged during the integrationperiod.

Reference is now made to FIG. 8, which shows a top view of an example ICthat has a multiplexed array of individually-addressableprobe-functionalized NMEs. By individually addressable, it is meant thateach NME may be individually electrically accessed, such that thecurrent or charge generated by each NME may be individually measured. Inthis example, the NMEs are arrayed in a row-column fashion. There are nrows and m columns for a total of m×n independent NMEs. If there wereonly a single row or column of NME, then it may not be necessary to haverow/column address circuitry. However, when large total numbers of NMEsare desired on a single device, it may be more efficient to array themin a two-dimensional grid or similar, and thus independent electricalaccess to each NME may be useful. This may be efficiently achieved usingthe circuitry illustrated in FIG. 7. In this approach, the chargeassociated with the current flowing through each NME is integrated intoa charge storage, such as a capacitor; and a voltage proportional to thestored charge may be read out for the NME in a particular row by settingthe node SEL (20) to a high level and monitoring the voltage on thatcolumn V_(col).

The figure illustrates that, for each column, there may exist atime-dependent signal (whose time-dependence may be determined by theclocking of the row-address circuitry) which, in some embodiments, maybe fed, in cases with the aid of electronic buffering or amplification,into an analog-to-digital converter. The analog-to-digital converter mayaccept signals having a pre-determined voltage swing (such as 0-1 V,typically) and, for each input channel, may carry out a quantizationoperation in which a digital representation of the analog level in thatsignal is estimated. The output of the A/D converter is a digital streamwhich combines parallelism (e.g., multiple parallel wires, eachcorresponding to a significant figure in the binary representation ofthe values) and serial timing (e.g., a timed representation ofsequential data elements corresponding, for example, to differentprobe-functionalized NMEs).

Reference is now made to FIG. 9, showing three adjacent NMEs along asingle row, in a configuration that may be provided on a biosensingdevice or IC as described above. These three NMEs are read using threedifferent columns j, j+1, and j+2. This figure illustrates a number offeatures with respect to the differences among NMEs.

NMEs E_(i,j) and E_(i,j+1) may be functionalized both with the sameclass of probes (e.g. thiolfunctionalized PNA), but the sequences may bedifferent. That is, each NME may be functionalized with similar probesthat have different target biomolecules. In this example, electrodesE_(i,j) and E_(i,j+1) are response to different sequences present in thesample under study. In sum, the use of different fimctionalizationsenables sensing of biomolecules within a single class, but having adifferent sequence, conformation, or functionality.

NMEs E_(i,j), E_(i,j+1), and E_(i,j+2) are shown having differentmorphologies and/or sizes, and different degrees of nanostructuring. Asdiscussed above, different morphologies and/or sizes may provide bothdifferent limits of detection, and different dynamic ranges, indetecting of target molecules. By incorporating NMEs having differentmorphologies and/or degrees of nano structuring onto one device, it maybe possible to expand the dynamic range of target concentrations thatmay be sensed using a single device. In sum, the use of different NMEmorphologies, sizes and/or nanostructurings may enable sensing of awider range of concentrations of a given target species than wouldotherwise be achieved if only one morphology/nanostructuring wereprovided on a biosensing device.

NMEs E_(i,j+1) and E_(i,j+2) are also depicted as being functionalizedusing different classes of probe molecules. For example, E_(i,j+1) maybe functionalized using a nucleic acid such as PNA, and E_(i,j+2) may befunctionalized using antibodies which attach to the electrode. In sum,the use of different classes of probe molecules may enable sensing ofdifferent classes of target biomolecules, for example ranging from DNAto RNA to micro-RNA to proteins, using a single biosensing device.

Biosensing Cartridge and Workstation

The biosensing device as described above may be incorporated into abiosensing cartridge. Such a cartridge may contain chambers for sampleprocessing such as disruption and resin or bead-based nucleic acidpurification, as well as a chamber for the biosensing device. Thecartridge may be self-contained, for example all necessary reagents maybe contained in the lid of the cartridge. The cartridge may be reusable,or may be disposable. A disposable cartridge may minimize the risk ofcross-contamination between samples.

The cartridge may be used in a biosensing workstation for coordinatingand carrying out the biosensing process. Components of the workstationmay include sample holders, instrument tips such as pipettors formanipulation of the sample, a sample identification module, a selectionmechanism for selecting a test to be carried out, an electronic displayfor indicating the results of a biosensing test and a processor formanaging these components and carrying out the selected tests. Theworkstation may hold a number of different cartridges at one time (e.g.,ten or more). The workstation may allow random access to thecartridges—that is, independent tests may be run at any time on anycartridge in the workstation. The workstation may have disposableinstrument tips, which would be the only part of the workstation thatcomes into direct contact with the sample and reagents. Disposable tips,together with disposable cartridges, may minimize the risk ofcross-contamination between samples tested in the workstation.

In general, a biosensing cartridge may have a first chamber forcontaining the sample to be tested, and a second chamber containing thebiosensing device as described above. There may be addition chambers toperform other actions on the sample, such as purification, and/orsubdivision (e.g., through chemical, mechanical or vibrational means).Some processing and disruption of the sample may be carried out in thefirst chamber itself. The sample may be introduced from the first to thesecond chamber for detection by the biosensing device in the secondchamber. There may be pre-set time interval from activation of thedevice or start of the test to the introduction of the sample into thesecond chamber. This time interval may allow the biosensing device to besuitably biased or otherwise readied for the test. The charge or currentflow generated in the biosensing device may be measured after anintegration period, as described above.

Reference is now made to FIG. 10, showing an example of a biosensingcartridge and the steps of using such a cartridge. In this example, thecartridge has three chambers, a sample chamber containing the sample, apurifying chamber for purifying the sample, and a biosensing forperforming the biosensing. As shown in this example, the lid of thecartridge is provided with capsules containing reagents for eachchamber, and through which an instrument tip may be inserted. Thisallows the cartridge to be self-contained, already containing thereagents suitable to carry out the biosensing operation and tailored tothe particular probes and/or target biomolecules of the biosensingdevice being used. In this example, the sample chamber has one capsule,for accessing the sample. The purifying chamber has three capsules—twocontaining a washing reagent, and one containing an elution buffer. Thebiosensing chamber has three capsules—each containing an electrochemicalmix containing analytes for the biosensing device. The instrument tipmay be inserted sequentially in each capsule, in order to carry out thebiosensing operation. For example, the capsule on the sample chamber maycontain a lysis buffer containing chemical denaturants (e.g., urea orformamide); the capsules one the purifying chamber may have two capsulescontaining a wash buffer and the elution buffer may be a standardbuffer, such as one containing low levels of sodium, chloride and trissalt; the capsules on the biosensing chamber may contain redox reportergroups such as ruthenium hexamine, ferricyanide and a buffer containingsodium, phosphate, chloride and magnesium.

In use, the sample is first extracted from the sample chamber throughits single capsule. The sample is then introduced into the purifyingchamber, where it is washed twice as the instrument tip is introducedthrough the two washing capsules and the elution buffer is introduced.Through this process, the sample may be prepared for biosensing by thebiosensing device. For example, in the case of a nucleic acid sample,the process in the purifying chamber may isolate the RNA or DNA in thesample. The purified sample is then introduced into the biosensingchamber, through each electrochemical capsule. The biosensing device inthe biosensing chamber may then detect any target biomolecules presentin the sample, and the generated current or charge may be measured.Where the cartridge is used in a workstation, the processor in theworkstation may read this generated current or charge and determine thepresence of the target biomolecule based on this reading.

Reference is now made to FIG. 11, showing an example of a biosensingworkstation. This work station includes a bar code reader, allowingidentification of samples using unique bar codes provided on eachcartridge. The workstation has a selection mechanism, in this example atouch screen that allows the selection of a particular test to becarried out. There is also a waste container for disposing any wastesgenerated by the biosensing process. The processor of the workstationmay be connected to an external computing device, such as anotherworkstation, for further analysis. This connection may be through awireless network. The workstation may be relatively small (e.g., afootprint of 1.5×1 ft), allowing convenience and ease of use.

Methods of Use

Methods for using NMEs and devices comprising the same is now described.A device may be provided with the NME already functionalized with aprobe molecule, or the probe molecule may be bound to the NME whenpreparing the device for use. The device is then biased for use, forexample by adding an electrocatalytic reporter and waiting a certaintime interval. In addition to the NME, there may be a referenceelectrode, which may or may not be an NME, in contact with theelectrocatalytic reporter but not in contact with the sample. Thecurrent flow or voltage bias generated over this time interval betweenthe NME and the reference electrode may be measured and recorded as thereference point. The NME is then exposed to a sample of interest, andthe current flow or charge generated over a certain time interval (alsoreferred to as the integration period) may be measured. By comparing thedifference in current flow or charge between the exposure time intervaland the biasing time interval, the concentration, binding and/or amountof target biomolecule in the sample may be determined.

Devices comprising NMEs, as described herein, may be used in conjunctionwith appropriate probes to detect the presence or absence of particularbiomarkers in a sample. A “sample” or “biological sample” as hereinrefers to any natural (e.g. plant, animal, algal, bacterial or viral) orsynthetic material containing DNA, RNA and/or proteins, including, forexample, clinical samples, such as tissues, cell cultures or fluidsisolated from an individual (including without limitation blood, plasma,serum, cerebrospinal fluid, lymph, tears, urine, saliva, mucus, synovialfluid, cerebrospinal fluid and tissue sections) environment (e.g.,water, food or air samples). Biological samples may be further processedvia a variety of means, including lysis (electrical, mechanical andchemical), electrophoresis, enzymatic digestion. Most often, the samplehas been removed from an organism, but the term “biological sample” canalso refer to cells or tissue analyzed in vivo, i.e., without removal.Typically, a “biological sample” will contain cells, but the term canalso refer to non-cellular biological material, such as non-cellularfractions of blood, saliva, or urine. “A biological sample” furtherrefers to a medium, such as a nutrient broth or gel in which an organismhas been propagated, which contains cellular components, such asproteins or nucleic acid molecules.

Probes for use with the instant described NMEs may be comprised ofnucleic acids. A “nucleic acid probe” refers to a nucleic acid (e.g. aribonucleic acid (RNA), deoxyribonucleic acid (DNA) or an analogthereof, including, for example, a peptide nucleic acid (PNA), whichcontains a backbone comprised of N-(2-aminoethyl)-glycine units linkedby peptides rather than deoxyribose or ribose linked byphosphodiesterase linkages) capable of binding to a target nucleic acidof complementary sequence through one or more types of chemical bonds,usually through complementary base pairing, usually through hydrogenbond formation. As used herein, a nucleic acid probe may include natural(i.e., A, G, C, or T) or modified on bases (7-deazaguanosine, inosine,etc.) or on sugar moiety. In addition, the bases in a probe can bejoined by a linkage other than a phosphodiester bond, so long as it doesnot interfere with hybridization. It will be understood by one of skillin the art that probes may bind target sequences lacking completecomplementarity with the probe sequence depending upon the stringency ofthe hybridization conditions. By assaying for the presence or absence ofthe probe, one can detect the presence or absence of the select sequenceor subsequence. Methods for detecting target nucleic acids using nucleicacid probes are described, for example, in U.S. Pat. No. 7,361,470entitled “Electrocatalytic Nucleic Acid Hybridization Detection.” and US2005/0084881 of the same name.

“Hybridization” refers to any process by which a strand of nucleic acidbinds with a complementary strand through base pairing. “Hybridizationconditions” refer to standard conditions under which nucleic acidmolecules are used to identify similar nucleic acid molecules. Suchstandard conditions are disclosed, for example, in Sambrook et al.,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Labs Press,1989. Sambrook et al., ibid., is incorporated by reference herein in itsentirety (see specifically, pages 9.31-9.62). In addition, formulae tocalculate the appropriate hybridization and wash conditions to achievehybridization permitting varying degrees of mismatch of nucleotides aredisclosed, for example, in Meinkoth et al., 1984, Anal. Biochem. 138,267-284; Meinkoth et al., ibid., is incorporated by reference herein inits entirety. Non-limiting examples of hybridization conditions includelow stringency hybridization conditions, moderate stringencyhybridization conditions and high stringency hybridization conditions.

In another embodiment, the probe is a peptide (comprised of, forexample, 4-40 amino acids) or proteins (e.g. antibody) that is able tobind to or otherwise interact with a biomarker target (e.g. receptor orligand) to provide an indication of the presence of the ligand orreceptor in the sample. Methods for detecting analytes using peptide orprotein probes are described, for example in International patentapplication WO 2007/094805 (PCT/US2006/013771) entitled “Method forElectrocatalytic Protein Detection.”

Probes may include a functional group (e.g., thiol, dithiol, amine,carboxylic acid) that facilitates binding with an NME. Probes may alsocontain other features, such as longitudinal spacers, double-strandedand/or single-stranded regions, polyT linkers, double stranded duplexesas rigid linkers and PEG spacers.

As described above, the surface nanostructure of the NME may becontrolled, and may influence the sensitivity and/or efficiency of adevice having the NME. In Example 1, the influence of surfacenanostructure on the detection efficiency for nucleic acids wasinvestigated. Two different types of NMEs were compared—a more finelynano structured NME obtained with a low deposition potential wascompared to a more coarsely textured one obtained with a higherdeposition potential.

While concentrations as low as 1 pM could be detected with the morefinely nanostructured NME obtained with a low deposition potential, thedetection limit was increased to 10 pM for the more coarsely texturedone obtained with a higher deposition potential. These resultsdemonstrate that increased nanostructuring contribute to more sensitivebiosensing capabilities in an electrode platform. This analysis revealedthat the more finely structured NMEs showed greater responsiveness tosub-nM concentrations of target sequences.

The 10 aM sensitivity observed here with the disclosed NMEs andelectrocataytic reporter system provides a low detection limit for alabel- and PCR-free sensor; the detection limit corresponds to thedetection of <100 copies of the target sequence. While the measurementof 60-1000 copies of target sequence has been achieved previously withelectrochemical detectors exploiting multi-step catalytic readout (Mungeet al., Anal. Chem. 77:4662, Nicewamer-Pena et al., Science 294:137,Park et al., Science 295:1503, Sinensky et al., Nat. Nano. 2:653,Steemers et al., Nat. Biotechnol. 18:91, Xiao et al., J. Am. Chem. Soc.129:11896, Zhang et al., Nat. Nano. 1:214, Zhang et al., Anal. Chem.76:4093), the disclosed device provides this measurement on a chip-basedplatform with single-step readout.

Example 2 describes use of a multiplexed electrode platform, asdescribed herein, to directly read a panel of cancer biomarkers inclinically-relevant samples using electronic signals. The systemcombines nanotextured electrodes with rapid catalytic readout to achievea long-standing goal: the multiplexed analysis of cancer biomarkersusing an inexpensive and practical platform.

Example 3 describes the use of an NME based chip to detect microRNA, oneof the most challenging detection targets. Electronic readout ofmicroRNA profiles offers a rapid, yet highly accurate, method todirectly assay RNA samples for specific sequences without the need fortarget amplification.

Although the provided examples are directed to the detection of cancerbiomarkers, other applications may be possible for the NME device, whichmay involve detecting DNA, RNA and/or proteins. Examples includeprofiling of breast cancer genes (e.g., by detecting RNA markers);profiling of leukemia-related genes (e.g., by detecting RNA markers);profiling of cytochrome P450 mutations that affect drug metabolism (e.g.Warfarin) (e.g., by detecting DNA and RNA markers); profiling ofmutations associated with genetic disease (e.g. Cystic fibrosis) (e.g.,by detecting DNA markers); detection and typing of viruses (e.g. HPV andHIV) (e.g., by detecting DNA and RNA markers); detection ofcancer-related proteins using an electrochemical immunoassay format(e.g. prostate specific antigen (PSA)) (e.g., by detecting proteinmarkers); and detection of micro RNAs to identify cancer. Biosensingdevices incorporating these NMEs may be adapted to detect for theseother biomolecules by binding suitable probes to the NME and/or byselecting a suitable electrocatalytic reaction to be sensed, as commonlyknown in the art.

It would be understood by a person skilled in the art that variationsare possible without departing from the present disclosure. All examplesand embodiments described are provided for the purpose of illustrationonly, and are not intended to be limiting. All references mentionedherein are hereby incorporated by reference in their entirety.

Example 1 Parameters for Manufacturing NMEs

In this example, Pd was used as an electrode material. To investigatethe time-dependence of the electrodeposition, the structures of the PdNMEs being electrodeposited were monitored as a function of time.Time-dependent electrodeposition experiments were performed at −100 mVusing 0.5 M HCl as supporting electrolyte. Pd structures were formed for(a) 25, (b) 50, (c) 125, (d) 250, and (e) 500 s. After 50 s, structureswith average diameters of 1.3 μm and heights of 0.5 μm were observed,and after 500 s the Pd electrodes were typically 8 μm and 5 μm indiameter and height. The smaller structures made with shorter depositiontimes typically exhibited depressions in the center of themicroelectrodes, which may indicate that nucleation occurspreferentially at the boundary of the aperture.

Another controllable parameter that may influence the final structure ofthe NME is the deposition potential. Specifically, the size and surfacemorphology of the NMEs may be controlled in this way. Dendritic fractalsare phenomena generally observed in nonequilibrium growth such as thegrowth of snowflakes, the aggregation of soot particles, and thesolidification of metals. Such fractal structures are also obtained bynonequilibrium electrodeposition of metals and used as model systems forthe study of branching and fractal growth processes (Fleury, Nature1997, 390, 145-148). It is generally believed that the morphology ofcrystals heavily depends on the “distance” of their formation conditionsfrom the thermodynamic equilibrium: near-equilibrium conditions lead topolyhedral crystals surrounded with thermodynamically stable crystalfaces, but increase of this “distance” makes the growing fronts ofcrystals with flat surfaces instable to form dendrites (Fukami et al. J.Phys. Chem. C 2007, 111, 1150-1160). In the case of electrodeposition ofmetals, such “distance” may be tuned continuously and reversibly bysimply changing the deposition potential and more negative potential canexert higher driving force and thus increase the “distance” from theequilibrium for electrocrystallization. Thus, electrodeposition may becontrolled spatially and kinetically to produce arrayed NMEs with variedwell-defined morphologies.

Pd structures were formed at (a) 0 mV, (b) −100 mV, (c) −250 mV, and (d)−400 mV for 250 s with the use of 0.5 M HCl as supporting electrolyte.More negative deposition potentials were found to typically lead tolarger, but less compact, microstructures. At a deposition potential of0 mV, a cake-shaped structure of 3.5 μm diameter and 0.7 μm height wasobtained. When the applied potential was changed to −100 mV, a roughermicro structure was obtained that was also larger in size (averagediameter=5 μm and height=2.5 μm). The nanotexturing obtained is anirregular aggregate of very small nanoparticles. When a more negativepotential of −250 mV was applied, a dendritic fractal micro structurewas obtained and its diameter and height were found to further increase11 and 6 μm, respectively. If the potential was increased further (e.g.to −400 mV), the microelectrode structure became more open and thestructure is no longer continuous.

The electrochemical behaviour of the Pd NMEs formed as a function ofpotential was studied by cyclic voltammetry (CV). The electrochemicalresponse of the NMEs was monitored in a solution containing 3 mMRu(NH₃)₆ ₃₊ and 0.09 M sodium phosphate, at a scan rate of 100 mV/s. Asexpected, steady-state voltammograms are observed for each electrode,consistent with the microscale dimensions of the electrodes. For theelectrodes made using deposition potentials of 0, −100, or −250 mV, thecurrents observed are well-correlated with the size of the electrode.That is, the greater the diameter of the NME (i.e., formed at a largerapplied potential), the greater the response current observed. However,for structures made at −400 mV, currents observed were lower thanexpected based on the size of the microelectrode, indicating that thediscontinuity of the electrode structure may lead to poor electricalconnectivity and loss of working area.

Thus, moderate deposition potentials appear to provide the mostpronounced nanotexturing while maintaining the integrity of theresultant NME, as small nanoparticles are formed on the surface of themicroelectrodes. It appears that providing too large of a drivingpotential for the deposition reaction accelerates the kinetics to apoint where metal nanoparticles are formed without strong connectivityto the core of the NME.

NME morphology may also be controlled via electrolyte effects. Pd NMEswere formed at −100 mV for 250 s using (a) 0.5M H₂SO₄ and (b) 0.5M HClO₄as the supporting electrolyte. These structures were formed under thesame conditions as described above, where HCl was used as a supportingelectrolyte. The structures formed in H₂SO₄ and HClO₄ were significantlylarger than those formed in HCl, and interestingly, all three displayeddifferent types of nanostructuring. NMEs made in HClO₄ showed the finestnanostructuring, with features as small as 10-20 nm present. In HCl, theelectrode was more compact, and the nano structuring was on the order of100 nm. The coarsest nanostructuring was obtained in H₂SO₄, where theparticles comprising the electrode were larger than 200 nm. Theseobservations indicate that the NME morphology may also be controlled byvarying the supporting electrolyte used for electrodeposition.

Typically, the dendritic structures for the NME depend on the conditionsduring manufacture, including concentrations of the electrodepositionsolution, choice of the metal to be electrodeposited, and the appliedpotential during electrodeposition. These parameters are readilycontrollable. For example, it may be desirable to control theconcentration and purity of the reagents used in electrodeposition towithin 5%. The choice of metal is simple to control as long as purity ofthe reagents is high, simply by obtaining the correct material. Thepotential during electrodeposition may be readily controlled to within afew mV, which is sufficient for controlling the size and morphology ofthe resultant NME.

Example 2 Direct Profiling of Prostate Cancer Biomarkers in Tumor TissueUsing a Multiplexed Nanostructured Microelectrode Integrated Circuit

Materials and Methods

Chip Fabrication.

The chips were fabricated at the Canadian Photonics Fabrication Center.3″ silicon wafers were passivated using a thick layer of thermally grownsilicon dioxide. A 350 nm gold layer was deposited on the chip usingelectron-beam assisted gold evaporation. The gold film was patternedusing standard photolithography and a lift-off process. A 500 nm layerof insulating silicon dioxide was deposited using chemical vapordeposition. 500 nm apertures were imprinted on the electrodes usingstandard photolithography, and 2 mm×2 mm bond pads were exposed usingstandard photolithography.

Fabrication of Nanostructured Microelectrodes.

Chips were cleaned by rinsing in acetone, IP A, and DI water for 30 sand dried with a flow of nitrogen. All electrodeposition was performedat room temperature with a Bioanalytical Systems Epsilon potentiostatwith a three-electrode system featuring an Ag/AgCl reference electrodeand a platinum wire auxiliary electrode. 500 nm apertures on thefabricated electrodes were used as the working electrode and werecontacted using the exposed bond pads. Platinum NMEs were fabricated ina platinum bath containing 5 mM solution of H2PtC16 and 0.5 M HClO₄ at−250 mV for 10 s using DC potential amperometry.

Preparation and Purification of Oligonucleotides.

All synthetic oligonucleotides were stringently purified byreversed-phase HPLC. The following probe and target sequences were usedin experiments. Seq. P1. Type III fusion probe (PNA): NH2-Cys-Gly-ATAAGG CTT CCT GCC GCG CT-CONH2 (SEQ ID NO. 1), Seq. P2. Type I fusionprobe (PNA): NH2-Cys-Gly-CTG GAA TAA CCT GCC GCG CT-CONH2 (SEQ ID NO.2), Seq. P3. Type VI fusion probe (PNA): NH2-Cys-Gly-ATA AGG CTT CTG AGTTCA AA-CONH2 (SEQ ID NO. 3), Seq. T1 (Type III TMPRSS2:ERG fusion DNAtarget): 5′AGC GCG GCA GGA AGC CTT AT3′ (SEQ ID NO. 4), Seq. T2 (WTTMPRSS2 DNA target): 5′AGC GCG GCA GGT CAT 10 ATT GA3′ (SEQ ID NO. 5),Seq. T3 (WT ERG DNA target): 5′TCA TAT CAA GGA AGC CTT AT3′ (SEQ ID NO.6), Seq. T4 (noncomplementary DNA target): 5′TTT TTT TTT TTT TTT TTTTT3′ (SEQ ID NO. 7). Oligonucleotides were quantitated by measuringabsorbance at 260 nm and ext. coefficients calculated using:http://www.idtdna.com/analyzer/Applications/OligoAnalyzer/.

Modification of NMEs with PNA Probes.

A solution containing 500 nM thiolated single stranded PNA, 25 mM sodiumphosphate (pH 7), and 25 mM sodium chloride was heated at 50oC for 10minutes. A suitable amount of 10 mM MCH was then added to make the finalMCH concentration of 100 nM. 0.5-10 μL (depending on the degree ofmultiplexing) of this mixture was deposited on the NMEs in a darkhumidity chamber overnight at 4° C. The NMEs were rinsed in 25 mM sodiumphosphate (pH 7), and 25 mM NaCl buffer before measurement.

Electrochemical Measurements.

Electrochemical signals were measured in solutions containing 10 μMRu(NH3)6 3+, 25 mM sodium phosphate (pH 7), 25 mM sodium chloride, and 4mM Fe(CN)6 3−. Differential pulse voltammetry (DPV) signals before andafter hybridization were measured using a potential step of 5 mV, pulseamplitude of 50 mV, pulse width of 50 ms, and a pulse period of 100 ms.Cyclic voltammetry signals before and after hybridization were collectedwith a scan rate of 100 mV/s. Limiting reductive current (I) wasquantified by subtracting the background at 0 mV from the cathodiccurrent at −300 mV in a cyclic voltammetry signal. Signal changescorresponding to hybridization were calculated as follows:ΔI=(Ids−Iss)/Iss×100% (ss=before hybridization, ds=after hybridization).

Hybridization Protocol.

Hybridization solutions typically contained target sequences in 25 mMsodium phosphate (pH 7), and 25 mM NaCl. Electrodes were incubated at37° C. in humidity chamber in dark for 60 minutes and were washedextensively with buffer before electrochemical analysis.

Isolation of mRNA.

The mRNAs were extracted from cell lines and patient tissue samples withthe Dynabeads mRNA Direct Kit (Invitrogen). Two typical prostate cancertissue samples were obtained from radical prostatectomies collected byfrom the Cooperative Human Tissue Network. The tissue was stored at −85°C. until tumor-rich tissue was selected for mRNA extraction. Theconcentrations of mRNA targets were measured by NanoDrop ND-1000 ofThermo Fisher Scientific (USA). All of the fusion sequences wereconfirmed by RT-PCR and direct sequencing.

Kinetic Measurements of DNA Hybridization at NMEs.

PNA (seq. 2)-modified NMEs were prepared as described above. Rinsed NMEswere immersed in a solution containing 10 μM Ru(NH3)6 3+, 4 mM Fe(CN)63−, 100 fM DNA target (seq. 4 to 7), 25 mM sodium phosphate (pH 7), and25 mM NaCl. The electrocatalytic CV signals were obtained as describedabove. All measurements were performed at 37° C.

Results and Discussions

We sought to generate a nanomaterial-based platform for ultrasensitivebioanalysis that is i) highly robust and straightforward to fabricate;ii) multiplexed and scalable; and iii) sensitive and specific whenpresented with heterogeneous biological samples. To satisfy requirementsi) and ii) we required a means of achieving reproducible placement ofeach individual sensing element using a scalable protocol. To addressrequirement iii), we sought to incorporate nanoscale features into oursensing array. The production of arrayed nanostructured sensingelements, however, can be labor-intensive and prone to lowreproducibility. Electron-beam lithography provides the needed controlover nanoscale features and their placement; however, it is a serialtechnique not presently suited to low-cost, high-volume chip production.Our approach was instead to use cost-effective conventionalphotolithography to position and address our electrodes; and then find ameans to bring about, with a high degree of reproducibility, thenanostructuring of these microelectrodes.

We constructed an 8-fold multiplexed chip by patterning a 350 nm thickgold layer on a silicon chip to create eight 5-μm-wide Au wires attachedto large metal pads that would serve as external contacts. SiO₂ was thendeposited as a passivating layer and patterned to create apertures with500 nm diameters at the end of each of the Au wires. These openings werecreated to serve as individual templates for controlled, local growth ofnanostructures. We then used palladium electrodeposition to depositmetal in the patterned apertures. We found that we were able to regulatethe size of the nanostructures by varying the deposition time. We werereadily able to confine the diameter of the structures to theultramicroelectrode regime (<10 u). Under conditions enabling rapidmetal deposition, the surfaces of the microelectrodes displayed a highlevel of nano structuring, with feature sizes of approximately 20 nm.These structures displayed ideal microelectrode behavior, exhibiting lowcapacitive currents and high steady-state plateau currents.

In order to make these nanostructured microelectrodes (NMEs) functionalas nucleic acids biosensors, we modified them with thiolatedpeptide-nucleic acids (PNA) probes. The use of PNA as a probe moleculehas been shown previously to increase the sensitivity of biosensingassays and is particularly advantageous in electrochemical assaysbecause it produces lowered background currents. To transduce nucleicacids hybridization into an electrical signal, we employed anelectrocatalytic reporter system previously developed by our laboratory.(Lapierre, M. A. et al., Anal. Chem. 2003, 75. 6327-6333). This reportersystem relies on the accumulation of Ru(NH3)63+ at electrode surfaceswhen polyanionic species like nucleic acids bind, and the catalysis ofthe reduction of Ru(III) via the inclusion of Fe(CN)63−, whichregenerates Ru(III) and allows multiple reductions per metal center.When PNA-modified NMEs were challenged with a complementary sequence,detectable signal changes could be clearly detected through thefemtomolar concentration range. Negligible signal changes were observedwith completely non-complementary sequences.

The cancer biomarkers selected for analysis on this platform are a groupof gene fusions specific to prostate cancer. These fusions, resultingfrom a chromosomal translocation that joins the ERG and TMPRSS2 genes,were recently discovered and appear in at least 50% of prostate tumours.Furthermore, there are ˜20 sequence types that feature different fusionsites, and the exact type of fusion present in a tumour appears tocorrelate with its aggressiveness and metastatic potential. Thesesequences are therefore not only promising diagnostic markers, but arealso factors with prognostic value.

To determine whether the NME sensors could discriminate gene fusionsequences from the wild-type sequences that would be half-complementary,a sensor modified with a probe complementary to the splice site of theType III fusion was challenged with: (1) the fusion target (seq. T1),(2) the sequence corresponding to the wild-type TMPRSS2 gene (seq. T2),and (3) a sequence corresponding to the wild-type ERG gene (seq. T3). Acompletely non-complementary control was also assayed (seq. T4). With ahybridization time of 60 minutes, large signal increases were observedwith the fully complementary target, while a much lower signal changewas seen with the TMPRSS2 target. The ERG target produced an even lowersignal change, and that observed with the non-complementary sequence wasnegligible. The TMPRSS2 target binds to the portion of the probe locatedat the end of the sequence not attached to the electrode, while the ERGtarget binds to the portion of the probe located at the end tethered tothe electrode surface. The different signal levels observed indicatethat the most accessible side of the probe is better able to bindincoming target molecules, while hybridization with the more buried partof the sequence is inefficient.

To determine whether the hybridization of the different targets requiredthe full 60 minute time period originally tested for accurate readout,the electrocatalytic signals were monitored at a variety of intervalswithin the window originally tested. Interestingly, the rise of thesignals is very fast, with significant current changes observed within 2minutes. Over the total 60-minute period, however, the signals for thehalf-complementary and non-complementary sequences fall noticeably; with20-50% of the 2-minute signal vanishing by 60 minutes. It appears thatfor sequences that are not fully complementary, some nonspecific bindingoccurs in the first few minutes of exposure of the NME sensor to thetarget solution, but these complexes do not remain stable and do notremain immobilized on the electrode. Thus, while non-complementarysequences can be discriminated from complementary sequences with shorthybridization times, longer times increase the differential signalchanges, and thus the degree of specificity.

The performance of these nanostructured microelectrodes as nucleic acidsdetectors indicated that the patterned structures were indeed sensitiveand specific when used under appropriate hybridization conditions. Wetherefore sought to prove that multiplexed chip-based NMEs could be usedto assay cancer biomarkers presented in heterogeneous biologicalsamples. To explore this capability, cell extracts and tumour samplesfrom prostate cancer patients were assessed to determine whether thesensitivity and specificity of the system was robust enough for clinicaltesting.

To determine whether we could detect prostate-cancer associated genefusions using the NME chip, we first analyzed mRNA isolated from twoprostate cancer cell lines: VCaP and DU145. The former cell line is typeIII fusion positive, and the latter is fusion negative. No appreciablesignal changes occurred when 10 ng of mRNA from the cell line that lacksthis sequence were incubated with a NME displaying a probe complementaryto the type III fusion (seq. PI), while large signal increases wereobserved in the presence of 10 ng mRNA from the cell line that doescontain the type III fusion. In addition, the modification of NMEs witha probe complementary to a different fusion (seq. P2) did not yield asignificant signal with positive mRNA sample. The detection of the fusedgene is therefore highly specific. These results are significant, asefficiency in the use of sample (10 ng) and the total time required foranalysis (less than 1.5 hours) significantly improve upon otherdetection methods like fluorescence in situ hybridization (FISH) andsequencing.

The ultimate application of the NME chip is the direct, multiplexedanalysis of a panel of cancer biomarkers in relevant patient samples. Totest the performance of our device for this type of application, weanalyzed a panel of mRNA samples collected from cell lines and clinicaltumor samples for a series of gene fusions. We obtained a group ofsamples that would allow the detection of the three most common types ofprostate-cancer gene fusions: type I, type III, and type VI. Differentclinical outcomes are associated with these sequences, with type IIIfusions being the most common but correlating with low cancer recurrencerates, whereas type I and VI fusions are correlated with aggressivecancers with high levels of recurrence. It is therefore of greatinterest to be able to differentiate these fusions in tumours, and amethod that would permit their presence or absence to be assessedquickly and straightforwardly would be of value in their further studyand validation as diagnostic biomarkers.

Probes complementary to each of the three fusions were deposited ontheir respective electrodes on NME chips, and 5 different mRNA sampleswere profiled for the presence of different gene fusions in amultiplexed format. Three cell lines were tested: VCap (type IIIpositive), 28 NCI-H660 (type III and VI positive)30, and DU145 (fusionnegative). 28 In addition, two tumour samples (tissues collected byradical prostatectomies) were tested, one that was positive for the typeI fusion, and one that was positive for the type III fusion, asconfirmed by conventional sequencing. In each case, all experiments tookless than 2 hours and required only 10 ng of mRNA. By analyzing theelectrochemical signals collected at NMEs displaying different probes,we ascertained the identity of fused genes present in each sample. Forexample, in the patient sample containing the type I fusion (as verifiedby sequencing), the current values observed at each probe-modified NMEdecreased in the following order: I>>>>III>VI. In the patient samplecontaining the type III fusion, the electronic signals again pointed tothe correct identity of the fusion with probe III>>>>I>VI. Theseresults, and those obtained with DU145, VCaP, and H660 cellular RNA,where electronic profiling correctly called the absence or presence ofgene fusions, indicate that NME chips are able to profile theseimportant biomarkers in complex samples and to distinguish biomarkerprofiles associated with different clinical outcomes.

The detection platform described here is not only specific, sensitive,and robust, it is also practical and scalable. The reproduciblefabrication method we chose is amenable to the production ofprobe-modified chips using the same photolithographic technologies inwidespread use in consumer electronics microchip fabrication; and onlysimple, inexpensive instrumentation is needed for readout. Microfluidicsare not required for automated analysis, as hybridization can beperformed and read out in a single reaction vessel. This systemrepresents an attractive alternative to PCR-based methods that aresensitive but difficult to automate in a clinical setting.

In sum, the new multiplexed electrode platform we describe here is thefirst to read directly a panel of cancer biomarkers inclinically-relevant samples using electronic signals. The array enablingthese measurements features microelectrodes that possess controllableand versatile nanotexturing essential for sensitivity. The systemcombines these nanotextured electrodes with rapid catalytic readout toachieve a long-standing goal: the multiplexed analysis of cancerbiomarkers using an inexpensive and practical platform.

Example 3 Direct, Electronic MicroRNA Detection Reveals DifferentialExpression Profiles in 30 Minutes

Materials and Methods

Materials.

6-mercapto-1-hexanol (97% MCH), hexaamine ruthenium chloride (99.9+%),potassium ferricyanide (99%), and palladium (II) chloride(99.9+%) werepurchased from Sigma-Aldrich Canada Ltd (Oakville, ON), perchloric acid(70%), acetone (ACS grade) and isopropyl alcohol (IPA, ACS grade) wereobtained from EMD (Gibbstown, N.J.). Thiolated PNA oligomers wereobtained from Biosynthesis Inc (Lewisville, Tex.) with HPLC purifiedgrade. PNA probes a Cys-Gly dipeptide at their N-terminus. Gly acts as aspacer, while Cys provides free thiol for immobilization on theelectrode surface. Synthetic microRNAs (5′ end phosphorylated and HPLCpurified) were obtained from Euro fins MWG Operon (Huntsville, Ala.).All PNA and RNA sequences are shown in table 51 provided in thesupporting information.

Chip Fabrication.

The chips were fabricated at the Canadian Photonics Fabrication Center.3″ silicon wafers were passivated using a thick layer of thermally grownsilicon dioxide. A 350 nm gold layer was deposited on the chip usingelectron-beam assisted gold evaporation. The gold film was patternedusing standard photolithography and a lift-off process. A 500 nm layerof insulating silicon dioxide was deposited using chemical vapordeposition. 500 nm apertures were imprinted on the electrodes usingstandard photolithography, and 2 mm×2 mm bond pads were exposed usingstandard photolithography.

Fabrication of Nanostructured Microelectrodes.

Chips were cleaned by rinsing in acetone, IPA, and DI water for 30 s anddried with a flow of nitrogen. All electrodeposition was performed atroom temperature with a Bioanalytical Systems Epsilon potentiostat witha three-electrode system featuring an Ag/AgCl reference electrode and aplatinum wire auxiliary electrode. 500 nm apertures on the fabricatedelectrodes were used as the working electrode and were contacted usingthe exposed bond pads. A 2 mm portion of the chip was immersed into theplating bath containing 5 mM palladium (II) chloride and 0.5 Mperchloric acid, and incubated for about 5 min prior to electroplating.The bond pads were kept free from solution. Pd NMEs were fabricatedusing DC potential amperometry at an applied potential of −100 mV for 6s.

Modification of NMEs with PNA Probes.

Single-stranded thiolated PNA probes were dissolved in a buffer solution(pH 7) containing 25 mM sodium phosphate and 25 mM sodium chloride at aconcentration of 500 nM. The solution was then heated at 50oC for 10minutes to fully dissolve the PNA molecules. A suitable amount of 10 mMMCH was then added to make the final MCH concentration of 100 nM. 10 μLof this mixture was quickly deposited on a chip displaying Pd NMEs usinga manual micropipettor. This PNA probe>solution covered chip was thenincubated in a dark humidity chamber overnight at 4oC. Theprobe-modified Pd NMEs were vigorously rinsed with the above buffersolution before measurements. For multiplexed experiments, chips witheight individually addressable leads were used.

Target Hybridization.

Hybridization solutions contained various concentrations of targets in25 mM sodium phosphate (pH 7.0) and 25 mM NaCl. Pd NMEs were incubatedwith 10 μL of target solution at 37° C. in a humidity chamber for 30mins to allow the immobilized probe molecules to hybridize with targetmolecules. The chip was then cooled and washed vigorously with bufferbefore the electrochemical analysis.

Electrochemical Measurements.

Electrochemical measurements were performed with an electrochemicalanalyzer (BASi, West Lafayette, USA) in a solution containing 10 mMRu(NH3)6 3+, 4 mM Fe(CN)6 3−, 25 mM sodium phosphate (pH 7.0) and 25 mMNaCl. Cyclic voltammetry (CV) was conducted before and after theaddition of target solutions at a scan rate of 100 mV/s. Differentialpulse voltammetry (DPV) was performed at a potential step of 5 mV, pulseamplitude of 50 mV, pulse width of 50 ms and a pulse i period of 100 ms.Cyclic voltammetry signals before and after hybridization were collectedwith a scan rate of 100 mV/s. Limiting reductive current (I) wasquantified by subtracting the background at 0 mV from the cathodiccurrent at −300 mV in a cyclic voltammetry signal. Signal changescorresponding to hybridization were calculated as follows:ΔI=(Ids−Iss)/Iss×100 (ss=before hybridization, ds=after hybridization).The detection limit was determined as the first concentration wherebackground (noncomplementary ΔI) subtracted signal was 2 times higherthan the standard deviation of 10 fM non-complementary control sample.

SEM Imaging.

HITACHI S-3400 SEM (Hitachi High Technologies America, Inc., Pleasanton,Calif.) was employed to study the morphology and dimension of theelectroplated¹ NMEs. The chip was affixed on a stainless steel SEM stubusing doublesided adhesive black carbon tape. The SEM image was acquiredusing the secondary electron mode at 20 kV.

RNA Extraction for PCR Analyses and Amplification Protocol.

Total RNA was extracted from cell lines with mirVana kit (Ambion). Thequality of samples was assessed by RT-PCR analysis of the endogenouscontrol RNU44 using Applied Biosystems TaqMan® microRNA Assay. Thisassay includes a reverse transcription (RT) step using the TaqMan®MicroRNA Reverse Transcription Kit (Applied Biosystems, CA, USA) whereina stemloop RT primer specifically hybridizes to a mir molecule and isthen reverse transcribed with a MultiScribe reverse transcriptase.Briefly, the reverse transcription mix includes 50 nM stem-loop RTprimers, 1×RT buffer, 0.25 mM each of dNTPs, 3.33 U/μL MultiScribereverse transcriptase, and 0.25 U/μl RNase inhibitor. The 7.5 μLreaction was then incubated in an Applied Biosystems 7900 Thermocyclerfor 30 minutes at 16° C., 30 minutes at 42° C., 5 minutes at 85° C. andthen held at 4° C. The RT products were subsequently amplified withsequence-specific primers (hsa-mir-21 primer 4373090, and hsa-mir-205primer 4373093 from Applied Biosystems) using the Applied Biosystems7900 HT Real-Time PCR system. The 10 μL PCR mix contains 0.67 μL RTproduct, 1×4 TaqMan® Universal PCR Master Mix, 0.2 ρM TaqMan® probe, 1.5ρM forward primer and 0.7 ρM reverse primer. The reactions wereincubated in a 384-well plate at 95° C. for 10 minutes followed by 40cycles of 95° C. for 15 seconds and 60° C. for 1 minute.

Cloning and Expression of Pre-miRNA-21.

cDNA was produced by reverse transcribing 500 ng total RNA from FaDucells using High-Capacity cDNA Reverse Transcription Kit (AppliedBiosystems) according to manufacturer's instructions. 50 ng of cDNA wereused as template for PCR amplification of the premiRNA-21 stem loop with1 ρM of the primers MIR-F (CCT ACC ATC GTG AC A TCT CCA TGG) and MIR-R(ATG AGA ACA TTG GAT ATG GAT GGT). The conditions for the PCR were: 95°C. for 2 min, followed by 40 cycles of 95° C. for 1 min, 55° C. for 1min, and 72° C. for 1 min, and a final extension step at 72° C. for 10min. The PCR product was gel purified (Gel extraction kit, Qiagen) andcloned in pCR4-TOPO vector (invitrogen). Colonies were picked and grownin LB medium. Plasmid DNA was purified with Plasmid Mini Kit (Qiagen),and checked for the correct product, orientation, and absence ofundesired mutations with sequencing (ACGT corporation). Plasmid DNA wasdigested with NotI which does not generate 3′ protruding ends thatproduce high background vector RNA during in vitro transcription. 1 μgof linearized plasmid DNA was used as a template for in vitrotranscription with AmpliScribe™ T3 High Yield Transcription Kit(Epicentre Biotechnologies). After the completion of reversetranscription, DNase I was added to digest the template DNA and RNA waspurified with PureLink Micro to Midi Total RNA Purification Kit(Invitrogen). Concentration of RNA was determined by reading theabsorbance at 260 nm.

Results and Discussions

We endeavored to develop a new method for microRNA profiling that wouldfeature the convenience of array-based analysis, but would augment thepower of such multiplexing with the exceptional sensitivity required toassay small biological samples for low abundance microRNAs. Given thatconventional fluorescence-based methods are insufficiently sensitive tomonitor hybridization of small numbers of molecules to surface—boundprobe sequences with simple instrumentation, we instead pursued anapproach that employed electronic readout.

In order to provide a platform for electronic microRNA detection, amultiplexed chip was prepared that featured an electrode patterngenerated by photolithography. This chip was made using a silicon waferas a base, and a pattern of gold was deposited on its surface to providea multiplexed set of leads and external contacts. A layer of SiO wasdeposited on top of the gold to passivate the metal, and then in thefinal fabrication step, 500 nanometer apertures were opened on the endof each lead to expose gold. To generate protruding micorelectrodes,palladium was electrodeposited in the apertures. The electrodepositionstep was engineered to produce highly nanostructured microelectrodes(NMEs). Previous studies have indicated that nano structured sensingelements can present biomolecular probes more efficiently than bulkmaterials and facilitate surface complexation reactions, but thisadvantage has never been exploited for direct biological profiling.

To test the electronic chip for sensitivity and specificity in microRNAdetection, Pd NMEs were modified with PNA probes and exposed to RNA forhybridization. Complexation was assayed using a redox reporter system,previously shown to exhibit femtomolar sensitivity when used inconjunction with nanostructured electrodes and PNA probes. (R. Gasparac,et. al., J. Am. Chem. Soc. 2004, 126, 12270; Z. Fang, S. 0. Kelley,Anal. Chem. 2009, 81, 612; M. A. Lapierre, et al., Anal. Chem. 2003, 75,6327; M. A. Lapierre-Devlin, et al., Nano Lett. 2005, 5, 1051). Thisreporter system relies on the accumulation of Ru(III) when nucleic acidshybridize at an electrode surface, and the signals obtained from thisreporter are amplified by the inclusion of ferricyanide, which canregenerate Ru(III) chemically after its electrochemical reduction.Titrations of the miR-21 sequence showed detectable signal changesrelative to non-complementary control sequences when solutionscontaining as little as 10 aM of the target were exposed to thechip-based NMEs. This corresponds to 10 molecules per microliter ofsample. The very high level of sensitivity is accompanied by a limiteddynamic range of only 10², but for the detection of microRNAs, thistradeoff is merited given the low abundance of these sequences.

Two crucial additional sensing criteria are specifically demanded inmicroRNA detection. First, closely related sequences—different by as fewas one base—must be accurately distinguished. Second, sequenceappendages like those found in mature vs. precursor microRNAs, must bediscriminated. We sought to challenge our system with each of theserequirements. We investigated first the specificity of the assay formature microRNA sequences. This was conducted by analyzing signalchanges observed when the chip was exposed to solutions containingeither the full-length, double-stranded, precursor form of miR-21, orthe significantly shorter, single-stranded, mature miR-21 sequence. Thesignal obtained for the hairpin precursor structure approachedbackground levels, while a robust signal change was observed for maturemiR-21.

We evaluated the sensitivity of the detection approach to pointmutations by monitoring the response of probe-modified sensing elementsto two closely related sequences, miR-26a and miR-26b. Probescomplementary to each sequence were arrayed on the chip, and theresponse of these elements to the complementary sequences was monitored.The signal obtained when miR-26a was introduced was approximately 4times for the fully matched miR-26a probe over the mismatched miR-26bprobe, and similarly, the signal obtained when miR-26b was introducedwas approximately 4.5-fold higher for the fully matched miR-26b probeover its mismatched counterpart probe. These results indicate that thismultiplexed chip can successfully discriminate closely related microRNAsequences.

Deriving a “fingerprint” of microRNA expression from cell linesrepresenting a particular tumour type relative to normal cells has beenpreviously shown to be a powerful approach to identify microRNAs thatcan serve as biomarkers in patients. Having confirmed the specificityand sensitivity of the chip towards microRNA targets, we then tested itusing RNA samples extracted from human normal cells and those derivedfrom human head and neck squamous cancer cell lines grown in culture.For example, total RNA extracted from the human hypopharyngeal squamouscancer FaDu cell line and a normal oral epithelial cell line wastitrated onto a nanostructured microelectrode displaying a probecomplementary to miR-205. A positive signal was obtained with as littleas 5 ng of RNA derived from the FaDu cells, while normal epithelialcells did not produce any signal change with up to 20 ng of RNA. Thisindicates that the signal response corresponds to a unique markerpresent at significantly higher levels in the cancer cell lines.

We profiled two different microRNAs, miR-21 and miR-205, and alsoincluded a control RNA, RNU-44 in a panel of total RNA samples. Weemployed three different head and neck squamous cancer cell lines, andcompared the response of the microelectrode chip to these total RNAsamples relative to RNA isolated from normal oral epithelial cells. Asexpected, RNU-44 levels, as judged by the electrochemical responsemeasured for each total RNA sample exposed to a sensing element modifiedwith a complementary probe, remained constant in all four cell lines.However, miR-21 and miR-205 signals were both significantly elevated inthe cancer cell lines. Indeed, the levels of these microRNAs were judgedto be present at >100-fold higher levels in the cancer cell linesrelative to the normal epithelial cells. The over-expression of thesetargets was confirmed using conventional quantitative PCR (seesupporting information). Both miR-21 and miR-205 have been previouslyobserved to be elevated in primary human head and neck squamouscarcinomas, indicating a significant potential for these micro-RNAs toserve as diagnostic biomarkers for this malignancy.

In conclusion, the microRNA detection chip described here offers thesensitivity and specificity for the analysis of a novel class of nucleicacids biomarkers representing one of the most challenging detectiontargets. Electronic readout of microRNA profiles offers a rapid—yethighly accurate—method to directly assay RNA samples for specificsequences, and the lack of labeling or amplification renders thisapproach to be extremely straightforward and efficient, features notattainable with other PCR or hybridization-based approaches.

1-43. (canceled)
 44. A biosensing device comprising: a substrate; atleast one electrically conductive lead on the substrate; an insulatinglayer covering the lead, the insulating layer having an apertureexposing a portion of the lead; a nanostructured microelectrode adaptedby means of an electrocatalytic reporter system to generate a charge inresponse to a biomolecular stimulus, wherein the nanostructuredmicroelectrode is fractal; at least one probe molecule attached to saidmicroelectrode; and wherein said microelectrode is in electricalcommunication with the exposed portion of the lead.
 45. The biosensingdevice of claim 44, wherein the lead comprises a material selected fromthe group consisting of: Au, Al, W, TiN, and polysilicon.
 46. Thebiosensing device of claim 44, wherein the substrate comprises amaterial selected from the group consisting of: silicon, silica, quartz,glass, sapphire, gallium arsenide, germanium, silicon carbide, indiumcompounds, selenium sulfide, ceramic, plastic, polycarbonate and otherpolymer or combinations thereof.
 47. The biosensing device of claim 44,wherein the insulating layer is comprised of a material selected fromsilicon dioxide, silicon nitride, nitrogen doped silicon oxide, andparylene or combinations thereof.
 48. The biosensing device of claim 44,wherein a plurality of microelectrodes is provided in an array, and eachmicroelectrode is individually electronically accessible.
 49. Thebiosensing device of claim 44, wherein the probe is selected from thegroup comprising: nucleic acids, peptide nucleic acids, locked nucleicacids, proteins or peptides functionalized with suitable tetheringmolecules; and wherein the biomolecular stimulus is nucleic acidhybridization or protein-to-protein binding.
 50. A method of carryingout a biosensing process using a biosensing device comprising ananostructured microelectrode, wherein the nanostructured microelectrodeis spiky or fractal and includes a probe attached thereto, said methodcomprising: biasing the microelectrode relative to a referenceelectrode; measuring a reference charge or reference current flowbetween the microelectrode and the reference electrode; exposing themicroelectrode to a biomolecular stimulus; measuring by means of anelectrocatalytic reporter system, a charge or current flow generated atthe microelectrode in response to the biomolecular stimulus binding tothe probe; and determining the amount of biomolecular stimulus presentby comparing the measured charge or measured current flow against thereference charge or reference current flow.
 51. A biosensing cartridgecomprising: a sample chamber comprising a biological sample; abiosensing chamber containing a biosensing device comprising ananostructured microelectrode, wherein the nanostructured microelectrodeis spiky or fractal.
 52. The biosensing cartridge of claim 51, whereinthe biosensing chamber comprises a buffer comprising sodium, phosphate,chloride, magnesium or combinations thereof.
 53. The biosensingcartridge of claim 51, further comprises a purifying chamber forpurifying or isolating the sample.
 54. A method of detecting abiomolecular stimulus using a biosensing device comprising ananostructured microelectrode, wherein said nanostructuredmicroelectrode is spiky or fractal and includes a probe attachedthereto, said method comprising: biasing the microelectrode relative toa reference electrode; measuring a reference charge or reference currentflow between the microelectrode and the reference electrode; exposingthe microelectrode to a biological sample; measuring by means of anelectrocatalytic reporter system a charge or current flow generated atthe microelectrode in response to the biomolecular stimulus in saidbiological sample binding to the probe; and determining the amount ofbiomolecular stimulus present by comparing the measured charge ormeasured current flow against the reference charge or reference currentflow.
 55. The method of claim 54, wherein the probe is selected from thegroup comprising: nucleic acids, peptide nucleic acids, locked nucleicacids, proteins or peptides functionalized with suitable tetheringmolecules;
 56. The method of claim 54, wherein the biomolecular stimulusis nucleic acid hybridization or protein-to-protein binding.
 57. Themethod of claim 54, wherein the microelectrode is comprised of amaterial selected from the group consisting of: a noble metal, an alloyof a noble metal, a conducting polymer, a metal oxide, a metal silicide,a metal nitride, carbon or a combination of any of the same.
 58. Themethod of claim 54, wherein the biological sample comprises a tissuesample, cell culture isolate, blood, plasma, serum, cerebrospinal fluid,lymph, tears, urine, saliva mucus, or combinations thereof.
 59. A methodof detecting the presence of a biomarker in a biological sample using abiosensing device comprising a nanostructured microelectrode adapted togenerate a charge in response to a hybridization or protein-to-proteininteraction event, wherein said nanostructured microelectrode is spikyor fractal and includes a probe attached thereto capable of hybridizingor binding to the biomarker of interest, the method comprising: biasingthe microelectrode relative to a reference electrode; measuring areference charge or reference current flow between the microelectrodeand the reference electrode; exposing the microelectrode to thebiological sample; measuring by means of an electrocatalytic reportersystem, a charge or current flow generated at the microelectrode inresponse to the hybridization or interaction of the probe with thebiomarker of interest, wherein the presence of a charge or current flowas compared to the reference is indicative of the presence of thebiomarker of interest.
 60. The method of claim 59, wherein the probe isselected from the group comprising: nucleic acids, peptide nucleicacids, locked nucleic acids, proteins or peptides functionalized withsuitable tethering molecules.
 61. The method of claim 59, wherein themicroelectrode is comprised of a material selected from the groupconsisting of: a noble metal, an alloy of a noble metal, a conductingpolymer, a metal oxide, a metal silicide, a metal nitride, carbon or acombination of any of the same.
 62. The method of claim 59, wherein thebiological sample comprises a tissue sample, cell culture isolate,blood, plasma, serum, cerebrospinal fluid, lymph, tears, urine, salivamucus, or combinations thereof.
 63. The method of claim 59, wherein thebiomarker of interest is a gene over-expressed in cancer cells.
 64. Themethod of claim 59, wherein the biomarker of interest is a viral gene.